WO2005083084 described intercalator pseudonucleotides capable of being incorporated into the backbone of an oligonucleotide or an oligonucleotide analogue. Oligonucleotides comprising the intercalator pseudonucleotides have a reduced capability of triplex formation, but have the ability to discriminate between DNA and RNA, i.e. they form more stable complexes with DNA than with RNA.
Malakhov et al, 2004 (Eur. J. Org. Chem. 2004, 1298-1307) disclosed a monomer for incorporation into an oligonucleotide or an oligonucleotide. The aim of the study was to provide a natural base, i.e. a promiscuous base that can fit into a Watson-Crick helix opposite to any of the naturally occurring bases. No studies on triplex formation were reported.
The sequence-specific recognition of double-stranded DNA (dsDNA) is a topic of considerable interest in the development of oligonucleotide-based tools in molecular biology, therapeutics and bionanotechnology. Triple helixes are formed when a single-stranded triplex-forming oligonucleotide (TFO) binds to dsDNA through specific major groove interactions and this has been the subject of intense research for gene targeting. This approach allows transcriptional control, gene knock-out and sequence-selective treatment of genomic DNA aiming mutated or recombined genes.
The third strand affinity of TFOs to their targets is generally problematic due to their required recognition to homopurine sequences of dsDNA and the disfavored formation of pH sensitive C+•G-C Hoogsteen base triples at physiological conditions in the parallel (pyrimidine) binding motif. During the past decade, many efforts have been devoted to modify TFOs to improve binding affinity to their targets along with the design of triplex nucleobases which could alleviate restriction of the dsDNA sequence. Oligonucleotides possessing modified nucleic acids such as peptide nucleic acids (PNA), locked nucleic acids (LNA), 2′-aminoethyl-oligoribonucleotides (2′-AE-RNA) and N3′->P5′ phosphoramidates inducing increased binding affinity are among the most successful chemically modified TFOs. The stabilization of the triplex structures has been also observed upon addition of heterocyclic compounds (intercalators) sometimes possessing a positively charged side chain to the aqueous solution containing all three oligonucleotide sequences. It has been also shown that an intercalator covalently linked to the 3′- or the 5′-end of a TFO led to thermal stabilization of parallel triplexes in a range of +3.0-+16.1° C. depending on linker length and type of intercalator. However, there has been limited attention to the covalently attached intercalators inserted as a bulge in the middle of TFO.
This design could have several advantages. Firstly, the synthesis of only one phosphoramidite of intercalating pseudo-nucleotides is required compared to the synthesis of at least four nucleotide monomers needed for sugar modified nucleic acids. Secondly, several bulged insertions of an intercalator monomer into the sequence could considerably increase duplex and triplex stabilities compared to the single insertion. Moreover, the structural difference between Watson-Crick and Hoogsteen binding modes along with the absence or presence of 2′-OH in DNA and RNA give rise to different properties for the various types of helixes. Therefore, bulged insertions of a linker and breaking up the helix by intercalators are expected to result in unique properties for appropriately chosen helixes. This has led to chemically modified oligonucleotides which could discriminate between different types of single-stranded nucleic acids.